Prostaglandin D synthase-specific monoclonal antibody

ABSTRACT

The invention relates to a monoclonal antibody specifically recognizing lipocalin-type prostaglandin D synthase (L-PGDS), a hybridoma producing the monoclonal antibody, methods for detection of L-PGDS or diseases by the monoclonal antibody, and a kit for detection of L-PGDS by the monoclonal antibody. According to the invention, there is provided a monoclonal antibody specific to L-PGDS.

FIELD OF THE INVENTION

The present invention relates to a monoclonal antibody specific to humanL-PGDS present predominantly in cerebrospinal fluid (CSF), a hybridomaproducing said monoclonal antibody, methods for detection of L-PGDS ordiseases by said monoclonal antibody, and a kit for detection of L-PGDSby said monoclonal antibody.

BACKGROUND OF THE INVENTION

Prostaglandin D is a biologically active substance synthesized in animaltissues from arachidonic acid released from a biomembrane uponstimulation, and it is produced from prostaglandin H (a common precursorof prostaglandin families produced by cyclo-oxygenase) by prostaglandinD synthase (PGDS).

The presence of two types of PGDS, glutathione-independent L-PGDS andglutathione-dependent spleen type PGDS, is known (Shimizu et, al., J.Biol. Chem., 254, 5222-5228 (1979); Urade et al., J. Biol. Chem., 260,12410-12415 (1985); Christ-Hazelhof and Nugteren, Biochim. Biophys.Acta, 572, 43-51 (1979); Urade et al., J. Biol. Chem., 262, 3820-3825(1987). The former is known to be predominantly located in the centralnervous system such as brain, epididymis, spinal cord, retina, and innerear (Urade et al., J. Biol. Chem., 260, 12410-12415 (1985); Ueno et al.,J. Neurochem., 45, 483-489 (1985); Urade et al., J. Biol. Chem., 262,3820-3825 (1987); Goh et al., Biochim. Biophys. Acta, 921, 302-311(1987); and Tachibana et al., Proc. Natl. Acad. Sci. USA, 84, 7677-7680(1987)), and the latter is known to be distributed broadly in almost allperipheral organs including spleen, bone marrow, digestive organs,thymus, and skin (Ujihara et al., Arch. Biochem. Biophys., 260, 521-531(1988); and Ujihara et al., J. Invest., Dermatol., 90, 448-451 (1988)).

On the other hand, a protein called β-trace was observed to be presentspecifically in human CSF, but its physiological function remainedunrevealed (Causen, J. Proc. Soc. Exp. Biol. Med., 107, 170-172 (1961)).

A certain correlation between β-trace known as a protein specific to CSFand severe brain disorders or certain diseases (multiple sclerosis,brain tumors, Meckel's syndrome and paraproteinemia) was noted from theobservation that β-trace levels depend on such disorders (Ericsson etal., Neurology, 19, 606-610 (1969); Olsson et al., J. Neurol. Neurosurg.Psychiat. 37, 302-311 (1974); Link, J. Neurol. Sci., 16, 103-114 (1972);Whistsed and Penny, Clinica Chimica Acta, 50, 111-118 (1974); and Chemkeet al., Clinical Genetics, 11, 285-289 (1977)). However, the exactcorrelation between β-trace and such disorders could not be determinedbecause the physiological function of β-trace still remained unrevealedand because there was no tool available for determining the exact amount(concentration) of β-trace.

Recently, the nucleotide sequence of cDNA coding for L-PGDS was reported(Nagata et al., Proc. Natl. Acad. Sci. USA, 88, 4020-4024 (1991)), andproduction of L-PGDS by genetic recombination became feasible. TheL-PGDS thus produced was examined and its amino acid sequence wasestimated and compared with an N-terminal partial amino acid sequence ofhuman β-trace in searching for their homology (Kuruvilla et al., BrainResearch, 565, 337-340 (1991), Zahn et al., Neuroscience Letters, 154,93-95 (1993)) or with the amino acid sequence of purified human β-trace(Hoffmann et al., J. Neurochem., 61(2), 451-456 (1993)), and furtherimmunological examination was made using polyclonal antibodies (Watanabeet al., Biochem. Biophys. Res. Communication, 203, 1110-1116 (1994)).These studies revealed that β-trace was identical with L-PGDS.

Prostaglandin D occurring abundantly in the central nervous systemfunctions, in one physiological action, as a neuromodulator of severalcentral actions including sleep promotion. Prostaglandin D synthase isconsidered as a key enzyme for sleep-wake activities (Hayashi, FASEB J.,5, 2575-2581 (1991)), and it is believed that at least a part of theL-PGDS secreted from competent cells is accumulated in CSF (Watanabe etal., Biochem. Biophys. Res. Communication, 203, 1110-1116 (1994)).

Accordingly, the analysis of L-PGDS distribution etc. in the centralnervous system is useful for detection of diseases in the centralnervous system, and it is expected that L-PGDS levels in CSF or humorcan also be used as an indicator in early diagnosis and prognosticobservations for other diseases caused by abnormalities in the centralnervous system. It is further expected that L-PGDS (or β-trace) can beused for examination of a reproduction ability, diagnosis of fetalgrowth, etc. because this enzyme is distributed in such humors derivedfrom genital organs, as semen, oviduct fluid and amniotic fluid, aswell. For such applications, there is demand for antibodies specificallyrecognizing L-PGDS.

Nevertheless, such antibodies have still not been established with highspecificity to meet such demand.

DISCLOSURE OF THE INVENTION

The object of the present invention is to provide a monoclonal antibodyspecifically recognizing L-PGDS, a hybridoma producing said antibody,methods for detection of L-PGDS or diseases by said monoclonal antibody,and a kit for detection of L-PGDS by said monoclonal antibody.

As a result of their eager researches, the present inventors havesuccessfully obtained a monoclonal antibody specifically recognizingL-PGDS from a hybridoma prepared by cell fusion between myeloma cellsand antibody-producing cells from an animal immunized with L-PGDS whichis a major protein in human CSF, and they thereby arrived at the presentinvention.

That is, the present invention relates to a monoclonal antibodyspecifically recognizing L-PGDS. The subclass of such monoclonalantibody includes immunoglobulin G1 or G2.

Further, the present invention relates to a hybridoma producing saidmonoclonal antibody by cell fusion between myeloma cells andantibody-producing cells from an animal immunized with L-PGDS.

Further, the present invention relates to methods for detection ofL-PGDS or diseases by said monoclonal antibody. An example of suchdiseases is oligospermia.

Further, the present invention relates to a kit for detection of L-PGDS,which is selected from a reagent containing said monoclonal antibodylabeled with an enzyme and a substrate solution, and a reagentcontaining said monoclonal antibody obtained by biotination,enzyme-labeled avidin, and a substrate.

Further, the present invention relates to a method for detection ofdiseases by said kit for detection of L-PGDS. An example of suchdiseases is oligospermia.

Hereinafter, the present invention is described in detail.

1. Production of the Monoclonal Antibody

Production of the present monoclonal antibody against L-PGDS consists ofthe steps of:

(1) Preparation of an antigen;

(2) Immunization and preparation of antibody-producing cells;

(3) Establishment of an antibody-titration system;

(4) Cell fusion;

(5) Selecting and cloning hybridomas; and

(6) Isolation of the monoclonal antibody.

Hereinafter, each step is described.

(1) Preparation of an Antigen

L-PGDS can be produced in large amounts in a usual manner by E. coli andCHO cells etc. with its known cDNA (Nagata et al., Proc. Natl. Acad.Sci. USA, 88, 4020-4024 (1991)). In this production of L-PGDS, arecombinant DNA containing the cDNA for L-PGDS is constructed andtransformed into a microorganism, which is then cultured to produce theenzyme. The L-PGDS thus produced can be purified from the culture byconventional means.

Then, an immunogen is prepared by dissolving the resulting L-PGDS in abuffer and then adding an adjuvant to it. Examples of such adjuvants areFreund complete adjuvant, Freund incomplete adjuvant, BCG, Hunter'sTitermax (CytRx Corporation), key hole limpet hemocyanin-containing oil,etc., and any of them can be mixed.

(2) Immunization and Preparation of Antibody-producing Cells

The immunogen thus obtained is administrated as antigen into mammalssuch as horse, monkey, dog, pig, cow, goat, sheep, rabbit, guinea pig,hamster and mouse, or birds such as pigeon and chicken. In particular,mouse, rat, guinea pig, rabbit and goat are preferably used. Any of theknown immunization methods may be employed preferably using i.v., s.c.,or i.p. administration. Immunization intervals are not particularlylimited, and the immunogen is given 2 to 10 times, preferably 2 to 5times, preferably at intervals of several days to several weeks, morepreferably 1 to 3 weeks.

1 to 10 days preferably 2 to 5 days after the final immunization,antibody-producing cells are prepared from the animal. Examples of suchantibody-producing cells are spleen cells, lymph node cells, thymocytesand peripheral blood cells, and generally spleen cells are usedconventionally. In the case of mouse, 0.01 μg to 1,000 μg, preferably 1to 300 μg antigen is given per animal in one administration.

(3) Establishment of an Antibody-titration System

It is necessary to establish a system of measuring the antibody titer inserum from the immunized animal or in a culture supernatant from theantibody-producing cells, so that the immune response level of theimmunized animal can be confirmed and the desired hybridoma can beselected from the fusion cells. For example, the antibody can bedetected conventionally using known methods such as enzyme immunoassay(EIA), radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA)and fluorescence immunoassay (FIA). Although the present method fordetecting the antibody is not limited to the above methods, ELISA isconveniently used for easy operations. Usually, L-PGDS is put to eachwell of a 96-well plastic microtiter plate and left at room temperatureto immobilize the enzyme onto the well. Then, the unbound sites of theL-PGDS as antigen are blocked with calf serum albumin, fetal bovineserum, skim milk, or gelatin. Then, an antiserum diluted with phosphatebuffered saline (referred to hereinafter as PBS) or a culturesupernatant of the hybridoma is added to each well. Subsequently,commercial secondary antibody labeled with an enzyme or fluorescentcompound or with biotin is added to each well, followed by adding acoloration substrate. The coloration occurring can be determined in aphotometer or fluorometer to quantify the antibody against L-PGDS.

(4) Cell Fusion

The myeloma cells to be subjected to cell fusion with theantibody-producing cells are those derived from animal species such asmouse, rat, and human, easily available to those skilled in the art. Thecell line used is preferably drug-resistant, which upon fusion with theantibody-producing cells, is rendered survivable in a selection mediumsuch as HAT medium. A cell line resistant to 8-azaguanine is generallyused. Because such cell line lacks hypoxanthine-guaninephosphoribosyltransferase (HGPRT-), it can not grow inhypoxanthine-aminopterine-thymidine (HAT) medium. The cell line used ispreferably cells not secreting immunoglobulins.

Examples of such myeloma cells are mouse myeloma cell lines such asP3X63Ag8 (ATCC TIB-9; Nature, 256, 495-497 (1978)), P3X63Ag8U.1 (P3U1)(ATCC CRL-1580; Current Topics in Microbiology and Immunology, 81, 1-7(1978), P3X63Ag8.653 (ATCC TIB-18; European J. Immunology, 6, 511-519(1976)), and P2/NSI/1-Ag4-1 (ATCC CRL-1581; Nature, 276, 269-270(1978)); rat myeloma cell lines such as for example 210.RCY.Agl.2.3(Y3-Agl.2.3) (ATCC CRL-1631; Nature, 277, 131-133 (1979)); human myelomacell lines such as U-266-AR1 (Proc. Natl. Acad. Sci. USA, 77, 5429(1980)), GM 1500 (Nature, 288, 488 (1980)), and KR-4 (Proc. Natl. Acad.Sci. USA, 79, 6651 (1982)).

The antibody-producing cells are obtained from spleen cells, lymph nodecells, thymocytes, and peripheral blood cells. Briefly, theantibody-producing cells are prepared as follows: Tissues such asspleen, lymph node or thymus are excised or blood are collected from theimmunized animals. These tissues are disrupted and then suspended in abuffer such as PBS or in a medium such as DMEM, RPMI 1640 and E-RDF.This cell suspension is filtered through e.g. a #200-250 stainless meshand then centrifuged to give the desired antibody-producing cells.

Then, the antibody-producing cells are subjected to cell fusion withmyeloma cells.

Before cell fusion, myeloma cells suitable for antibody production areselected. 10⁶ to 10⁸ cells/ml antibody-producing cells are mixed with10⁶ to 10⁸ cells/ml myeloma cells at a ratio of from 1:1 to 1:10 in ananimal cell growth medium, e.g. Eagle's minimal essential medium (MEM),Dulbecco's modified Eagle's medium (DMEM), RPMI-1640 medium, or E-RDFmedium. To enhance cell fusion, these cells are mixed at a ratio of e.g.1:6 and then incubated for 1 to 15 minutes in the presence of a fusionenhancer in e.g. RPMI-1640 medium containing dimethylsulfoxide at atemperature of 30 to 37° C. This fusion enhancer may be polyethyleneglycol with an average molecular weight of 1,000 to 6,000, polyvinylalcohol or Sendai virus. Alternatively, the antibody-producing cells canbe subjected to cell fusion with myeloma cells by electric stimulation(e.g. electroporation) in a commercial cell fusion apparatus.

(5) Selection and Cloning of Hybridomas

After cell fusion, the cells are screened for desired hybridomas. Theselective growth of the fusion cells in a selection medium may be usedfor screening as follows: The cell suspension is diluted 5- to 10-foldwith e.g. E-RDF medium containing 15% fetal bovine serum and then put toeach well of a microtiter plate at about 10² to 10⁶ cells/well, followedby addition of a selection medium (e.g. HAT medium) to each well.Thereafter, the cells are incubated while the selection medium isexchanged with fresh one at suitable intervals.

Where myeloma cells are from an 8-azaguanine-resistant strain and HATmedium is used as selection medium, the myeloma cells andantibody-producing cells, if they fail to fuse, will die during in vitroculture in about 7 days and antibody-producing cells in 10 days. Hence,hybridomas can be obtained from those cells beginning to grow after the10th day of culture.

The hybridomas are screened for the desired ones by examining theirsupernatants on the presence of the antibodies against L-PGDS. Thisscreening step can be carried out by any of the conventional methods.For example, a supernatant (first antibody) from hybridomas grown ineach well is put to a well with L-PGDS immobilized on it, then a labeledsecondary antibody is added to the well and incubated, and the bindingability of the secondary antibody is then examined in enzymeimmunoassays (EIA, ELISA), RIA, etc.

In more detail, the screening of hybridomas is carried out as follows:Natural or recombinant L-PGDS, which was used as immunogen, isimmobilized as antigen onto a 96-well microtiter plate. A culturesupernatant expected to contain the monoclonal antibody is added to eachwell and reacted with the immobilized antigen. Then, the antigen-boundmonoclonal antibody, if any, is reacted with another antibody(enzyme-labeled anti-immunoglobulin antibody). Alternatively, saidimmobilized monoclonal antibody is reacted with a biotinylatedanti-immunoglobulin antibody and then with enzyme-labeled avidin.Finally, each well is colored by adding an enzyme substrate solution.The hybridomas whose culture supernatants are colored in the wellshaving the immobilized natural or recombinant L-PGDS are those producingantibodies having the ability to bind to the L-PGDS.

These hybridomas can be cloned in conventional methods includinglimiting dilution, soft agar cloning, fibrin gel cloning andfluorescence excitation cell sorting to give the desired monoclonalantibody-producing hybridoma.

(6) Isolation of the Monoclonal Antibody

From the resulting hybridoma, the monoclonal antibody can be isolatedusing conventional methods such as cell culture method, ascitestransudate method, etc.

In the cell culture method, the hybridoma is cultured for 2 to 14 daysin a medium such as RPMI-1640, MEM, or E-RDF containing 10 to 20% calfserum or in a serum-free medium under conventional culture conditions,e.g. 37° C., 5% CO₂. The antibody can be obtained from the culture.

In the ascites transudate method, a mineral oil such as pristane(2,6,10,14-tetramethylpentadecane) is administrated by i.p. to the samemammal species as the mammal from which the myeloma cells were derived.Then, the hybridoma, 1×10⁷ to 1×10⁹ cells, preferably 5×10⁷ to 1×10⁸cells, are administrated by i.p. to the animal, and a large amount ofhybridoma cells are grown in the animal. After 1 to 4 weeks, preferably2 to 3 weeks, ascites fluid or serum is collected from the animal.

If it is necessary to purify the antibody from the ascites fluid orserum, it can be purified by conventional methods such as salting-outwith ammonium sulfate, ion-exchange chromatography on anion exchangere.g. DEAE cellulose, affinity chromatography on Protein A SEPHAROSE, andgel filtration, and these may be used singly or in combination.

2. Method of Detecting L-PGDS by the Monoclonal Antibody of the PresentInvention

The method of detecting L-PGDS according to the present invention can becarried out using said monoclonal antibody, as follows:

A 96-well microtiter plate is coated with a diluted sample such as CSF,serum etc. and then blocked with e.g. 0.2% gelatin in PBS. Then, themonoclonal antibody of the present invention, labeled with an enzyme, isadded to each well and then incubated; alternatively, the monoclonalantibody labeled with biotin is added to each well, then the plate iswashed, enzyme-labeled avidin or streptoavidin is added to each well,and the plate is further incubated. Then, the plate is washed and acoloration substrate such as ABTS(2,2′-azino-di-(3-ethyl-benzothiazoline-6-sulfonic acid)) is added toeach well. L-PGDS can be determined by examining this coloration in thecalorimetric method.

In another embodiment of the present invention, a 96-well microtiterplate is coated with the diluted monoclonal antibody of the presentinvention and then blocked with e.g. 0.2% gelatin in PBS. Then, adiluted sample such as CSF, serum etc. is added to each well and theplate is incubated. After washing the plate, another enzyme-labeledmonoclonal or polyclonal antibody solution is added to each well and theplate is incubated; alternatively, the monoclonal antibody or polyclonalantibody labeled with biotin is added to each well, then the plate iswashed, enzyme-labeled avidin or streptoavidin is added to each well,and the plate is further incubated. Then, the plate is washed and acoloration substrate such as ABTS(2,2′-azino-di-(3-ethyl-benzothiazoline-6-sulfonic acid)) is added toeach well. The L-PGDS can be determined by examining this coloration inthe calorimetric method. In this manner, it is possible to detect andquantify L-PGDS.

3. A Reagent for Measurement of L-PGDS by the Monoclonal Antibody of thePresent Invention

The monoclonal antibody of the present invention is useful as a reagentfor measurement of L-PGDS because it specifically binds to L-PGDS.Furthermore, the present monoclonal antibody is useful as a reagent indetermining the presence and distribution of not only L-PGDS as antigenbut also other similar antigens having the same epitope as that ofL-PGDS, as well as fragments of L-PGDS in biological samples such asorgans, tissues, cells and humor. Hence, the antibody of the presentinvention is useful as a reagent for such measurement and diagnosis. Thedetection or measurement of L-PGDS in organs, tissues, cells and humorcan be effected using quantitative or qualitative means such as EIA,ELISA, RIA, FIA, Western blot technique and immunohistochemistry, etc.

4. A Kit for Detection of L-PGDS

The kit of the present invention, if an enzyme is used as a label fordetection, contains the following ingredients:

(1) monoclonal antibody labeled with an enzyme; and

(2) substrate.

The kit of the present invention, if modified with the sandwich ELISAmethod, contains the following ingredients:

(1) monoclonal antibody;

(2) monoclonal or polyclonal antibody labeled with an enzyme; and

(3) substrate.

The kit of the present invention, if modified with the biotin-avidinmethod, contains the following ingredients:

(1) biotinated monoclonal antibody;

(2) enzyme-labeled avidin or streptavidin; and

(3) substrate.

The kit of the present invention, if modified with the sandwich ELISAand biotin-avidin methods, contains the following ingredients:

(1) monoclonal antibody;

(2) biotinated monoclonal or polyclonal antibody;

(3) enzyme-labeled avidin or streptavidin; and

(4) substrate.

In the ingredients, the “monoclonal antibody” means the monoclonalantibody of the present invention. The “polyclonal antibody” means anantibody contained in serum from an animal immunized with L-PGDS, and itcan be prepared in the following manner.

(1) Preparation of the Antigen

L-PGDS can be produced in large amounts in a usual manner by E. coli andCHO cells etc. with its known cDNA (Nagata et al., Proc. Natl. Acad.Sci. USA, 88, 4020-4024 (1991)). For production of L-PGDS, a recombinantDNA containing the cDNA for L-PGDS is constructed and transformed into amicroorganism, and the transformant is cultured to produce the enzyme.The resulting L-PGDS can be purified from the culture by conventionalmeans.

L-PGDS thus obtained is dissolved in a buffer, and an immunogen isprepared by adding an adjuvant to it. Examples of adjuvants are Freundcomplete adjuvant, Freund incomplete adjuvant, BCG, Hunter's Titermax(CytRx Corporation), key hole limpet hemocyanin-containing oil, etc.,and any of them can be mixed.

(2) Immunization and Preparation of Blood

The immunogen thus obtained is administrated into mammals such as horse,monkey, dog, pig, cow, goat, sheep, rabbit, guinea pig, hamster andmouse, or birds such as pigeon and chicken, among which mouse, rat,guinea pig, rabbit and goat are preferably used. Any of the knownimmunization methods can be employed preferably via i.v., s.c., or i.p.administration. Immunization intervals are not particularly limited, andthis administration is carried out 2 to 10 times, preferably 2 to 5times, at intervals of preferably several days to several weeks, morepreferably 1 to 3 weeks.

Antibody titer in blood from the immunized animal is determinedaccording to the above method in (3) “1. Production of the monoclonalantibody”. Blood samples found to have high antibody titer are left atroom temperature or 4° C. and centrifuged to give serum containing thepolyclonal antibody.

If it is necessary to purify the polyclonal antibody from the serum, itcan be purified by conventional methods such as salting-out withammonium sulfate, ion-exchange chromatography on anion exchanger e.g.DEAE cellulose, affinity chromatography on Protein A Sepharose, and gelfiltration separating molecules depending on molecular weight andstructure, and these may be used in singly or in combination.

According to the present invention, various diseases can be detected byuse of the present kit for detection of L-PGDS. For example,oligospermia can be diagnosed readily and rapidly by the present kitusing the monoclonal antibody.

The monoclonal antibody of the present invention can also be used topurify L-PGDS. That is, the monoclonal antibody of the present inventionis coupled in a usual manner to carriers such as agarose, cellulose,acrylamide gel, commercially available self-made affinity carriers andthen washed. L-PGDS can be purified easily with high yield by elutionfrom the column with a suitable solvent or buffer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph showing a profile in SDS-PAGE.

FIG. 2 is a photograph showing a profile in SDS-PAGE and Westernblotting of CSF and purified L-PGDS.

FIG. 3 is a photograph showing a profile in Western blotting of purifiedL-PGDS treated with N-glycanase.

FIG. 4A shows the result of an epitope mapping of each monoclonalantibody from Silverstrain.

FIG. 4B shows the result of an epitope mapping of each Polyclonalantibody.

FIG. 4C shows the result of an epitope mapping of the monoclonalantibody designated as 10A3.

FIG. 4D shows the result of an epitope mapping of the monoclonalantibody designated as 9A6.

FIG. 4E shows the result of an epitope mapping of the monoclonalantibody designated as 6B9.

FIG. 4F shows the result of an epitope mapping of the monoclonalantibody designated as 1B7.

FIG. 4G shows the result of an epitope mapping of the monoclonalantibody designated as 6F5.

FIG. 5 is a calibration curve for L-PGDS (by monoclonal antibody 1B7).

FIG. 6 is a calibration curve for L-PGDS (by monoclonal antibody 6F5).

FIG. 7 is a calibration curve for L-PGDS (by sandwich ELISA method usingmonoclonal antibody 1B7 as primary antibody and biotinylated polyclonalantibody as secondary antibody).

FIG. 8 is a calibration curve for L-PGDS (by sandwich-ELISA method usingmonoclonal antibody 6F5 as primary antibody and biotinylated polyclonalantibody as secondary antibody).

FIG. 9 is a calibration curve for L-PGDS (by sandwich ELISA method usingmonoclonal antibody 1B7 as primary antibody and biotinylated polyclonalantibody (A), biotinylated 7B5 (B) and biotinylated 10A3 (C) assecondary antibodies).

FIG. 10 is a calibration curve for L-PGDS (by sandwich ELISA methodusing monoclonal antibody 10A3 as primary antibody and biotinylatedpolyclonal antibody (A) and biotinylated 1B7 (B) as secondaryantibodies).

FIG. 11 is a calibration curve for L-PGDS (by sandwich ELISA methodusing monoclonal antibody 7F5 as primary antibody and biotinylatedpolyclonal antibody (A) and biotinylated 1B7 (B) as secondaryantibodies).

FIG. 12 is a drawing showing a calibration curve prepared using L-PGDSand calibration curves for L-PGDS which were prepared using severalsamples.

FIG. 13 is a photograph showing a profile in Western blotting.

FIG. 14 shows the result of measurement of L-PGDS.

FIG. 15 is a photograph showing a profile in SDS-PAGE of L-PGDS purifiedby monoclonal antibody.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention is described in more detail byreference to Examples. However, the present invention is not limited tothe Examples.

EXAMPLE 1 Production of the Monoclonal Antibody

(1) Preparation of the Antigen

L-PGDS was prepared as antigen by genetic engineering.

A GST gene fusion system (Pharmacia) was used for expression of theantigen in E. coli and purification. The following procedure was carriedout for fusion of L-PGDS with GST protein.

A 185 bp product was obtained by amplifying a region of cDNA coding forN-terminal region of L-PGDS by polymerase chain reaction (PCR) in thefollowing manner.

The primer nucleotide sequences used are:

Ec23ALA: Sequence ID NO:1.

78NMUTA: Sequence ID NO:2.

PCR was carried out using Taq DNA polymerase (Takara Shuzo Co., Ltd.),restriction enzymes EcoRI and XhoI, and T4 DNA ligase (Takara Shuzo)where 1 cycle reaction (94° C. for 5 seconds, 45° C. for 3 seconds, andat 72° C. for 5 seconds) was repeated 28 times.

With these primers, the partial nucleotide sequence between the 152 to327 positions (from guanine (G) to cytosine (C), corresponding to thepartial amino acid sequence between the N-terminal (alanine) and the 81position (serine) on the amino acid sequence of the mature proteinexcluding its signal sequence) was amplified and an EcoRI site wasintroduced into the 5′-terminal. Because this PCR product had an XhoIsite at the 238 position, the product was subcloned by digestion withrestriction enzymes EcoRI and XhoI. A recombinant DNA was obtained byreplacement, by the subcloned product, of the corresponding N-terminalregion of the cDNA for native L-PGDS (Nagata et al., Proc. Natl. Acad.Sci. USA, 88, 4020-4024 (1991)). This recombinant DNA was inserted intoan EcoRI site of vector pGEX-2T (Pharmacia) which was selected for GSTfusion protein.

Alternatively, a 521 bp product was obtained by amplifying that regionof cDNA coding for the whole mature protein of L-PGDS by PCR asdescribed below.

The primer nucleotide sequences used are:

forward primer: Sequence ID NO:3.

reverse primer: Sequence ID NO:4.

PCR was carried out using Taq DNA polymerase (Takara Shuzo) where 1cycle reaction (94° C. for 5 seconds, 45° C. for 3 seconds, and at 72°C. for 5 seconds) was repeated 28 times.

With these primers, the region between the 152 to 656 positions (fromguanine (G) to adenine (A), corresponding to,the region between theN-terminal (alanine) to the C-terminal (glutamine) of the amino acidsequence of the mature protein excluding its signal sequence) wasamplified, and a BamHI site was introduced at the 5′-terminal and anEcoRI site at the 3′-terminal. The amplified DNA was inserted intoEcoRI/BamHI sites of vector pGEX-2T (Pharmacia) for GST fusion protein.

The resulting expression vector for GST-L-PGDS fusion protein wastransformed in a usual manner into E. coli DH5 α or JM109. The fusionprotein produced by the transformant was recovered by selectiveabsorption onto affinity chromatography beads (Pharmacia) and subsequentelution with thrombin according to manufacture's instructions. In thismanner, about 2 mg L-PGDS was obtained from 100 ml culture of thetransformant.

Proteins produced by 2 independent clones (E. coli DH5α/pGDS2 and E.coli DH5α/pGDS7) were analyzed by SDS-PAGE on 10-20% gradient gel. Theresults are shown in FIG. 1.

In FIG. 1, lane 1 shows bands of molecular-weight markers; lanes 2 to 5,from one of the above clones; lanes 6 to 9, from the other clone; lanes2 and 6, homogenates of the respective clones; lanes 3 and 7, fractionsnot absorbed onto the affinity column; lanes 4 and 8, fractions elutedwith thrombin; and lanes 5 and 9, fractions eluted with a buffercontaining reduced glutathione. In lanes 2 and 6, a band correspondingto a molecular weight of about 45 kDa is the fusion protein, and thisband is scarcely observed in the GST-unbound fractions (lanes 3 and 7).The fractions eluted with glutathione (lanes 5 and 9) showed the fusionprotein band and GST band (molecular weight of about 25 kDa), which werenot eluted with thrombin.

According to these results in SDS-PAGE, L-PGDS was produced in E. coliin the form of an about 45 kDa fusion protein with GST, and the proteinwith a molecular weight of about 20 kDa, eluted with thrombin (lanes 4and 8), is L-PGDS itself because this molecular weight corresponds tothe molecular weight of 20 kDa deduced from the nucleotide sequence forL-PGDS.

(2) Preparation of the Antibody-producing Cells

A 0.5 ml solution containing 500 μg L-PGDS obtained in (1) was mixedwith 0.5 ml Freund complete adjuvant and emulsified for 3 to 5 minutes.As antigen, 100μl of the emulsion was administrated by s.c. into thetail rump of a BALB/c mouse. 3 weeks after the first immunization, thesame volume of another antigen emulsion in Freund incomplete adjuvantwas administrated by i.p. to the mouse for boosting. 3 weeks after thesecond immunization, 100 μg antigen (100μg antigen/200 μl PBS) wasadministrated to each mouse via its tail vein. 3 days after the finalimmunization, the spleen was excised from the immunized mouse anddisrupted in E-RDF medium to give a cell suspension.

(3) Cell Fusion

The suspended 1×10⁸ spleen cells were subject to cell fusion with 1×10⁷mouse myeloma cells P3-X63-Ag8-U1 (P3-U1) or P3-X-63-Ag8.653 in 50%(W/V) PEG (molecular weight 1,500, Boehringer Mannheim) according to themethod of Oi and Herzenberg (Selected

Methods in Cellular Immunology, 351-371, W. H. Freeman Co., USA press,1980).

(4) Selection of the Hybridoma

According to the above-mentioned method of Oi et al., the desiredhybridoma was selected in HAT medium, i.e. E-RDF medium containing 1.36mg/dl hypoxanthine, 19.1 μg/dl aminopterin, 387 μg/dl thymidine, 10%fetal bovine serum and 5% Origen HCF (IGEN).

(5) Selection of the Monoclonal Antibodies

The antibody-positive cells in 15 wells were cloned by repeatinglimiting dilution at least twice. The resulting 6 clones were culturedto give strains producing a significant amount of the monoclonalantibody specific to L-PGDS, as follows:

1×10⁷ hybridoma cells were cultured at 37° C. for 4 days in 5% CO₂ inthe 225 cm² flask containing 50 ml E-RDF medium with 10% fetal calfserum. Among the resulting 6 cell lines, 5 lines were selected. Theresults are shown in Table 1.

TABLE 1 number number number of of cell of antibody- number of antibodyAntigen total wells growth wells positive wells established wells hPGDS960 778 15 6 hPGDS: human lipocalin-type prostaglandin D synthase.

In Table 1, “number of cell growth wells” means the number of wellswhere hybridomas could grow in selective culture in HAT medium; “numberof antibody-positive wells”, the number of wells where antibodyproduction was detected by ELISA using the antigen prepared in Example 1(1); and “number of antibody established wells”, the number of wellswhere hybridomas producing the specific antibody were established bycloning. The 5 selected cell lines were designated 1B7, 6F5, 7F5, 9A6and 10A3, respectively. The antibodies produced by these cell lines weregiven the same designations as above. The cell lines 1B7, 6F5, 7F5, 9A6and 10A3 have been deposited as FERM BP-5709 (original, deposit date:Sept. 21, 1995), FERM BP-5710 (original deposit date: Sept. 21, 1995),FERM BP-5711 (original deposit date: Jun. 6, 1996), FERM BP-5712(original deposit date: Jun. 6, 1996) and FERM BP-5713 (original depositdate: Jun. 6, 1996), respectively, with National Institute of Bioscienceand Human-Technology, Agency of Industrial Science and Technology(Higashi 1-1-3, Tsukuba City, Ibaragi Pref., Japan).

(6) Production of the Monoclonal Antibodies

Each of the cell lines 1B7, 6F5, 7F5, 9A6 and 10A3, selected above in(5), was administrated by i.p. to mice. Remaining resulting cell strain,6B9 was also administrated by i.p. to mice. Briefly, 1 ml pristane wasadministrated by i.p. to each mouse. 2 weeks thereafter, 1×10⁸ hybridomacells were inoculated intraperitoneally the mouse, and 2 weeksthereafter, the transuded ascites was collected from the mouse.

The collected ascites was applied to Protein A affinity columnchromatography according to a conventional method.

As a result, 3 to 10 mg/ml antibodies against L-PGDS were obtained.

Typing of the antibodies of 1B7, 6F5 with Isotyping Kit (RPN29,AMERSHAM) indicated that all of them belong to IgGl subclass and possessλ light chain.

EXAMPLE 2 Properties of the Monoclonal Antibodies

(1) Specificity Examination by ELISA

A 96-well microtiter plate was coated with various types of L-PGDS andblocked with 0.2% gelatin in PBS. After blocking, each of the 6antibodies obtained in Example 1 (5 was examined for specificity inELISA after adding 50 μl antibody solution to each well. The results areshown in Table 2.

TABLE 2 antigen 1B7 6B9 6F5 7F5 9A6 10A3 E. coli antigen ◯ ◯ ◯ ◯ ◯ ◯ E.coli lysate X X X X X X CHO antigen ◯ ◯ ◯ ◯ X ◯ CSF antigen ◯ ◯ ◯ X X XO: Reactivity was observed.  X: No reactivity was observed.

The antigens in the table are as follows:

E. coli antigen, expressed in E. coli and purified by affinitychromatography;

E. coli lysate, carrying a vector not containing L-PGDS cDNA (negativecontrol);

CHO antigen, expressed in CHO cells and purified by a conventionalmethod; and

CSF antigen, purified from CSF by a conventional method.

The results in Table 2 suggested that at least 3 antibodies 1B7, 6B9 and6F5 specifically recognize L-PGDS itself.

(2) Specificity Examination by Western Blotting

The above antigens were electrophoresed by SDS-PAGE on 16% isocratic geland then subjected to Western blot analysis using 5 kinds of antibody.The results are shown in Table 3.

TABLE 3 antigens 1B7 6B9 6F5 9A6 10A3 E. coli antigen ◯ ◯ ◯ ◯ ◯ E. colilysate X X X X X CHO antigen ◯ ◯ ◯ X ◯ O: A signal was observed.  X: Nosignal was observed.

A signal, appearing in an immunoblot profile obtained in colorationafter Western blotting, was detected at a position corresponding to themolecular weight (about 20 to 30 kDa) of prostaglandin D synthase.Hence, the antigens showing such signals are considered specific toprostaglandin D synthase.

FIG. 2 shows the results of Western blot analysis using CSF antigen andCSF itself.

Each lane A shows CSF where 2 μl was applied. Each lane B shows L-PGDS(CSF antigen) purified from CSF where 50 ng was applied. Silver stainingindicated a large number of proteins in CSF, and the purified L-PGDSappears as a broad band with a molecular weight of about 27 kD.

Each monoclonal antibody was used in Western blotting. The resultsrevealed that all monoclonal antibodies (1B7, 6B9, 6F5, 7F5, 9A6 and10A3) are reactive exclusively to the single protein (lane A)corresponding to the purified L-PGDS (lane B), indicating that theseantibodies specifically recognize L-PGDS without reacting to othercontaminants in CSF.

In addition, the purified L-PGDS was treated with N-glycanase and thensubjected to Western blot analysis. The results are shown in FIG. 3.Each lane A shows the sample treated with glycanase, and each lane Bshows the sample not treated with glycanase. As shown in FIG. 3, thereis no significant difference in band density before and after treatmentwith glycanase in the case of 1B7, 6B9, 6F5, 7F5, and 1A3, while in thecase of 9A6, the intensity of the band was significantly increased aftertreatment with glycanase, suggesting that 9A6 recognizes a glycosylationsite or therearound.

Then, the isotype of each monoclonal antibody was determined using amouse monoclonal antibody typing kit (RPN29, Amersham). The results areshown in Table 4. Further, their Kd values toward the E. coli antigenand CSF antigen were determined by the method of Friguet et al. (J.Immunol. Methods, 77, 305-319 (1985)). The results are shown in Table 4.

TABLE 4 monoclonal immunoglobulin dissociation constant (nM) antibodysubclass E. coli antigen CSF antigen 1B7 IgG₁ (λ) 5.4 3.9 6B9 IgG₁(κ) >1000 >1000 6F5 IgG₁ (λ) 13.2 10.3 7F5 IgG₁ (κ) 0.65 4.1 9A6IgG_(2a) (κ)  7.42 >1000 10A3 IgG₁ (κ) 0.53 3.9

1B7, 6F5, 7F5 and 10A3 showed high affinities for the E. coli antigenand CSF antigen, indicating that these monoclonal antibodies recognizethe surface of native L-PGDS molecule. 9A6 showed a high affinity forthe E. coli antigen, but it showed least reactivity to the CSF antigen.The results, along with these results in FIG. 3, suggested that 9A6recognizes a glycosylation or therearound. 6B9 showed reactivity inWestern blotting as shown in FIG. 2, but showed no reactivity to the E.coli antigen or the CSF antigen, suggesting its recognition of thedenatured molecule of L-PGDS.

Then, deletion mutants of L-PGDS were prepared by truncation of itspartial N-terminal amino acid sequence step by step and used as antigensfor epitope mapping of their monoclonal antibodies. The PCR primers usedin this experiment are shown below and the results are shown in FIG. 4.The antigens used in this experiment were prepared in essentially thesame way as in Example 1(1) except that the L-PGDS product was recoveredin the form of a fusion protein with GST by boiling in 1% SDS, not bytreatment with thrombin.

forward primer A (amino acids 1 to 7): SEQ ID NO:3.

forward primer B (amino acids 7 to 12): SEQ ID NO:5.

forward primer C (amino acids 13 to 18): SEQ ID NO:6.

forward primer D (amino acids 30 to 35): SEQ ID NO:7.

forward primer E (amino acids 52 to 57): SEQ ID NO:8.

forward primer F (amino acids 68 to 73): SEQ ID NO:9.

forward primer G (amino acids 85 to 90): SEQ ID NO:1.

forward primer H (amino acids 99 to 105): SEQ ID NO:11.

forward primer I (amino acids 118 to 123): SEQ ID NO:12.

forward primer J (amino acids 134 to 139): SEQ ID NO:13.

forward primer K (amino acids 152 to 158): SEQ ID NO:14.

reverse primer (amino acids 163 to 168): SEQ ID NO:4.

FIG. 4 suggested that 7F5 and 10A3 recognize a sequence in Ala¹-Val⁶;9A6, Gln¹³-Asn²⁹; 6F5, Tyr⁸⁵-Val⁹⁸; and 1B7 and 6B9, Gly¹¹⁸-Pro¹³³

(3) Preparation of Calibration Curves

The above E. coli antigen was used for preparation of calibration curvesfor L-PGDS. The monoclonal antibodies used were 1B7 and 6F5. A 96-wellmicrotiter plate was coated with diluted E. coil antigen and blockedwith 0.2% gelatin in PBS. After blocking, the biotinylated monoclonalantibody 1B7 or 6F5 was added to each well and incubated. Then, theplate was washed, and a streptoavidin-horseradish peroxidase conjugatewas added to each well and incubated. The plate was washed, and acoloration substrate ABTS(2,2′-azino-di-(3-ethyl-benzothiazoline-6-sulfonic acid) was added toeach well. L-PGDS was determined by measuring this coloration by thecalorimetric method. The results are shown in FIGS. 5 and 6 for 1B7 and6F5, respectively.

Then, the sandwich ELISA method was used for calibration curves forL-PGDS. A 96-well microtiter plate was coated with diluted monoclonalantibody 1B7 or 6F5 and blocked with 0.2 % gelatin in PBS. Afterblocking, the diluted E. coli antigen was added to each well andincubated. Then, the plate was washed, and the biotinylated polyclonalantibody was added to each well and incubated. Then, the plate waswashed, and a streptoavidin-horseradish peroxidase conjugate was addedto each well and incubated. The plate was washed, and a colorationsubstrate ABTS (2,2′-azino-di-(3-ethyl-benzothiazoline-6-sulfonic acid)was added to each well. L-PGDS was determined by measuring thiscoloration by the colorimetric method. The results are shown in FIGS. 7and 8 for 1B7 and 6F5, respectively. As can be seen from FIGS. 5 to 8,about 10-fold higher sensitivity was attained using the sandwich ELISAmethod.

In another embodiment of the present invention, the sandwich ELISAmethod and the biotinylated polyclonal or monoclonal antibody were usedfor preparation of calibration curves. A 96-well microtiter plate wascoated with diluted monoclonal antibody 1B7, 10A3 or 7F5 as primaryantibody and blocked with 0.2% gelatin in PBS. The diluted E. coliantigen was then added to each well and incubated. Then, the plate waswashed, and the biotinylated polyclonal antibody or the biotinylatedmonoclonal antibody 1B7, 7F5 or 10A3 was added as secondary antibody toeach well and incubated. The plate was washed, and astreptoavidin-horseradish peroxidase conjugate was added to each welland incubated. The plate was washed, and a coloration substrate ABTS(2,2′-azino-di-(3-ethyl-benzothiazoline-6-sulfonic acid) was added toeach well. L-PGDS was determined by measuring this coloration by thecalorimetric method.

The results are shown in FIGS. 9 to 11.

FIG. 9 shows the calibration curves using monoclonal antibody 1B7 asprimary antibody, where curve A makes use of the biotinylated polyclonalantibody as secondary antibody; curve B, biotinylated 7F5 as secondaryantibody; and curve C, biotinylated 10A3 as secondary antibody.

FIG. 10 shows the resulting calibration curves using monoclonal antibody10A3 as primary antibody, where curve A makes use of the biotinylatedpolyclonal antibody as secondary antibody; and curve B, biotinylated 1B7as secondary antibody.

FIG. 11 shows the calibration curves using monoclonal antibody 7F5 asprimary antibody, where curve A makes use of the biotinylated polyclonalantibody as secondary antibody; and curve B, biotinylated 1B7 assecondary antibody.

As can be seen from FIGS. 9 to 11, high sensitivity can be attainedusing 10A3 or 7F5 as primary antibody and the biotinylated polyclonalantibody, or biotinylated monoclonal antibody 1B7, as secondaryantibody.

EXAMPLE 3 Detection of L-PGDS Derived From Various Human Tissues

(1) Detection of L-PGDS in CSF

A 96-well microtiter plate was coated with diluted monoclonal antibody1B7 or 6F5 and then blocked with 0.2% gelatin in PBS. Diluted human CSFwas then added to each well and incubated. Separately, the polyclonalantibody prepared using a rabbit as an immunized animal was purified byaffinity column chromatography on Protein A and then biotinylated withNHS-LC-Biotinylation Kit (PIERCE).

Subsequently, the above microtiter plate was washed, and thebiotinylated polyclonal antibody was added to each well and thenincubated. Subsequently, a streptoavidin-horseradish peroxidaseconjugate was added to each well and incubated. The plate was washed,and a coloration substrate ABTS(2,2′-azino-di-(3-ethyl-benzothiazoline-6-sulfonic acid) was added toeach well. L-PGDS was determined by measuring this coloration by thecalorimetric method. The results are shown in Table 5.

TABLE 5 Sample No. 1 2 3 4 5 L-PGDS (μg/ml) 23 31 20 10 18

(2) Detection of L-PGDS in blood

L-PGDS in human serum obtained by centrifugation of human blood wasdetermined by the same method as in (1) above except that diluted serumwas used. The results are shown in Table 6.

TABLE 6 Sample No. 1 2 3 4 5 L-PGDS (μg/ml) 0.45 0.29 0.21 0.41 0.26

(3) Detection of L-PGDS in amniotic fluid

L-PGDS in human amniotic fluid was determined by the same method as (1)above except that diluted amniotic fluid was used. The results are shownin Table 7.

TABLE 7 Sample No. 1 2 3 4 5 L-PGDS (μg/ml) 5.5 1.2 1.3 2.9 1.8

(4) Detection of L-PGDS in semen supernatant

L-PGDS in human seminal plasma was determined by the same method in (1)above except that diluted seminal plasma was used. The results are shownin Table 8.

TABLE 8 Sample No. 1 2 3 4 5 L-PGDS (μg/ml) 4.1 12 23 10 8.3

(5) Detection of L-PGDS in follicular fluid

L-PGDS in human follicular fluid was determined by the same method in(1) above except that diluted follicular fluid was used. The results areshown in Table 9.

TABLE 9 Sample No. 1 2 3 4 5 L-PGDS (μg/ml) 0.21 0.11 0.11 0.15 0.13

(6) Detection of L-PGDS in urine

L-PGDS in human urine was determined by the same method in (1) aboveexcept that diluted urine was used. The results are shown in Table 10.

TABLE 10 Sample No. 1 2 3 4 5 L-PGDS (μg/ml) 1.07 0.65 2.45 1.62 0.32

(7) Detection of L-PGDS in various humors

The sandwich ELISA system (7F5 monoclonal antibody×biotinylated 1B7monoclonal antibody) established in Example 2(3) was used forquantifying L-PGDS in human CSF, serum and urine. The results (means(±SE)) were compared with those of previous literatures. In this assay,TMBLUE (Intergen-CDP) was used as colouring substrate in place of ABTS.

The measurement means are shown in Table 11.A, and those of theliteratures are shown in Table 11.B. L-PGDS levels in amniotic fluid andseminal plasma were also determined in the same manner, and the results(means (±SE)) are shown in Table 11.A, as well.

TABLE 11.A sample number of samples means (±SE) (μg/ml) CSF 38 12.07 ±1.26  serum 12 0.27 ± 0.01 urine 10  1.56 ± 0.30* amniotic fluid 52 2.55± 0.22 seminal plasma 32 13.01 ± 1.72  *Excretion amount (mg) per day

TABLE 11.B number means sample of samples (±SD) (μg/ml) literature CSF12 40 Pepe, A. J. and Hochwald, G. M. (1967) Proc. Soc. Exp. Biol. 126,630-633 CSF 59 26(±6)  Link, H. and Olsson, J. E. (1972) Acta Neurol.Scadinav. 48, 57-68 CSF 35  27(±1.5) Olsson, J. E., Link, H., andMüller, R. (1976) J. Neurol. Sci. 27, 233-245 CSF 192  33(±11)Felgenhauer, K., Schädlich, H. J., and Nekic, M. (1987) Klin.Wochenschr. 65, 764-768 serum 25  3.9(±0.16) Olsson, J. E., Link, H.,and Nosslin, B. (1973) J. Neurochem. 21, 1153-1159 urine 15  3.6˜53.9*Whitsed, H. and Penny, R. (1974) Clin. Chim. Acta 50, 119-128 *Excretionamount (mg) per day (not mean).

As shown in the tables, the present means are lower than those of theliteratures, particularly with respect to serum considered to havecontaminants in abundance. This difference may result from the fact thatL-PGDS might be overestimated in the methods of the literatures becauseof their low specificity, and high specificity in the present assaysystem is clarified.

A typical calibration curve prepared in these measurements is shown inFIG. 12.

Curve A is a calibration curve where L-PGDS purified from CSF or therecombinant L-PGDS prepared in Example 1(1) was used as the standardsubstance. Curves B, C, D, E, and F are calibration curves preparedusing CSF, seminal plasma, urine, amniotic fluid, and serum,respectively. As can be seen from FIG. 12, the sample calibration curvesare nearly parallel with the calibration curve A, so L-PGDS can bespecifically determined in the present assay system without anyinfluence of contaminants in a sample.

Then, an additive test in the present assay system was carried out.Human CSF, serum, urine, and seminal plasma were diluted respectively toadjust their L-PGDS contents to about 20 ng/ml. Purified L-PGDS (CSFantigen) from CSF was added in an amount of 15 ng/ml to each sample. Theresults indicated that good recovery (98.5 to 104.6%) was obtained inevery sample as shown in Table 12.

TABLE 12 di- lution degree L-PGDS (ng/ml) recovery sample (-fold)initial conc. addition recovery (%) CSF 1 350  20.69 15.00 35.65 99.7CSF 2 600  21.17 15.00 36.31 100.9 serum 1 15 20.71 15.00 36.21 103.3serum 2 15 18.67 15.00 33.96 101.9 urine 1 40 20.86 15.00 36.55 104.6urine 2 40 18.56 15.00 33.34 98.5 seminal 500  16.82 15.00 31.70 99.2plasma 1 seminal 1000  19.51 15.00 34.93 102.8 plasma 2

EXAMPLE 4

L-PGDS in 5 samples (seminal plasma) was detected by Western blottingusing the monoclonal antibody 1B7. The results are shown in FIG. 13. Asshown in FIG. 13, L-PGDS was detected specifically, indicating that thismonoclonal antibody is reactive exclusively to L-PGDS in the presence ofother contaminants.

Then, the sandwich ELISA system (monoclonal antibody 1B7×biotinatedpolyclonal antibody) established in Example 2 was used to quantifyL-PGDS in 1 ml seminal plasma from each of healthy normal persons (40cases) and patients with oligospermia (10 cases). The number ofspermatozoa in 1 ml semen was determined with a Makler counting chamber.The results are shown in Table 13.

TABLE 13 L-PGDS number of spermatozoa (μg/ml) sample no. (×10⁴spermatozoa/ml) subject by ELISA  1   0 oligospermia patient 2.6  2  100oligospermia patient 0.8  3  100 oligospermia patient 1.0  4  800oligospermia patient 3.8  5  800 oligospermia patient 2.2  6 1000oligospermia patient 0.9  7 1800 oligospermia patient 1.0  8 2000oligospermia patient 5.0  9 2000 oligospermia patient 4.8 10 2000oligospermia patient 2.6 11 2500 healthy person 4.3 12 3000 healthyperson 7.5 13 3000 healthy person 23.6 14 3000 healthy person 30.0 153000 healthy person 9.5 16 3000 healthy person 2.5 17 3000 healthyperson 4.4 18 4000 healthy person 6.0 19 4000 healthy person 30.0 204200 healthy person 8.0 21 5000 healthy person 1.9 22 6000 healthyperson 42.0 23 6000 healthy person 1.5 24 6000 healthy person 3.8 256500 healthy person 2.0 26 7000 healthy person 3.2 27 7000 healthyperson 8.2 28 8000 healthy person 9.6 29 8000 healthy person 14.0 308500 healthy person 6.0 31 8800 healthy person 3.0 32 9000 healthyperson 8.0 33 9000 healthy person 7.1 34 10000  healthy person 10.1 3510000  healthy person 3.0 36 10000  healthy person 7.5 37 10000  healthyperson 6.0 38 11000  healthy person 0.3 39 12000  healthy person 17.0 4012000  healthy person 2.6 41 12000  healthy person 7.0 42 12000  healthyperson 1.1 43 13000  healthy person 10.5 44 13900  healthy person 30.045 15000  healthy person 16.0 46 15000  healthy person 5.1 47 15000 healthy person 3.5 48 16000  healthy person 13.0 49 17400  healthyperson 15.0 50 18000  healthy person 6.2

The results indicated that L-PGDS levels are 9.75±1.486 μg/ml healthypersons) and 2.470±0.509 μg/ml (oligospermia patients), and thedifference therebetween is statistically significant at P≦0.0008 level(Mann-Whitney method) or P≦0.0192 (Fisher method), in FIG. 14. Becausemany samples can be dealt with in a short time using the kit of thepresent invention, it is useful for diagnosis of oligospermia.

EXAMPLE 5

One-step purification of L-PGDS from CSF was attempted by coupling eachof monoclonal antibodies 1B7, 6F5, 9A6, 7F5 and 10A3 to carriers by useof an Affi-Gel Hz immunoaffinity kit (Bio-Rad).

First, 10 ml gel with each monoclonal antibody coupled thereto wasequilibrated with PBS. A CSF solution, prepared by diluting 10 ml of CSFat least 3-fold with PBS, was applied to the gel. The gel was washedwith 20 ml PBS containing 2 M NaCl, then 30 ml PBS containing 0.1%TRITON X-100, and finally with 50 ml PBS, and the protein was elutedwith 0.1 M sodium citrate (pH 3.0). Using any kind of antibody, L-PGDScan be efficiently adsorbed onto the gel and eluted with 0.1 M sodiumcitrate (pH 3.0), and the resulting preparation having enzymaticactivity was almost homologous in SDS-PAGE. The yield was about 80% andthe product was purified as high as 37-fold relative to the originalCSF.

One Example of such purification is shown in FIG. 15.

Industrial Applicability

According to the present invention, there is provided the monoclonalantibody specific to L-PGDS.

The analysis of the distribution of L-PGDS in the central nervous systemis useful for detection of diseases in the central nervous system, andit is expected that L-PGDS levels in CSF or humor can also be used as anindicator in early diagnosis and prognostic observations for otherdiseases caused by abnormalities in the central nervous system. It isfurther expected that L-PGDS (β-trace) can be used for examination of areproduction ability, diagnosis of fetal growth, etc. because thisenzyme is distributed in such humors derived from genital organs, assemen, oviduct fluid and amniotic fluid, as well. Further, it wasrecently revealed in our study that the gene for L-PGDS is expressed inthe heart too and therefore the distribution and levels of L-PGDS inblood and other humors can also be used for diagnosis of diseases in thecirculatory organs.

Accordingly, the monoclonal antibody provided according to the presentinvention is useful as a reagent in studying the expression, tissuedistribution, physiological action etc. of L-PGDS, and as a reagent forpathological diagnosis of various diseases in the central nervous systemas well as in the genital and circulatory organs.

14 1 22 DNA Artificial Sequence Description of Artificial SequenceSynthetic DNA 1 gggaattcat gcacccgagg cc 22 2 20 DNA Artificial SequenceDescription of Artificial Sequence Synthetic DNA 2 gaggtcaggg cgaagccacc20 3 29 DNA Artificial Sequence Description of Artificial SequenceSynthetic DNA 3 ccggatccgc acccgaggcc caggtctcc 29 4 30 DNA ArtificialSequence Description of Artificial Sequence Synthetic DNA 4 atgaattcactattgttccg tcatgcactt 30 5 26 DNA Artificial Sequence Description ofArtificial Sequence Synthetic DNA 5 ccggatcctc cgtgcagccc aacttc 26 6 26DNA Artificial Sequence Description of Artificial Sequence Synthetic DNA6 ccggatccca gccggacaag ttcctg 26 7 26 DNA Artificial SequenceDescription of Artificial Sequence Synthetic DNA 7 ccggatcctc gagctggctccaggag 26 8 26 DNA Artificial Sequence Description of ArtificialSequence Synthetic DNA 8 ccggatccga tggtggcttc aacctg 26 9 26 DNAArtificial Sequence Description of Artificial Sequence Synthetic DNA 9ccggatccga gacccgaacc atgctg 26 10 25 DNA Artificial SequenceDescription of Artificial Sequence Synthetic DNA 10 ccggatcctaccggagtccc cactg 25 11 27 DNA Artificial Sequence Description ofArtificial Sequence Synthetic DNA 11 ccggatccgt ggagactgac tacgacc 27 1226 DNA Artificial Sequence Description of Artificial Sequence SyntheticDNA 12 ccggatccgg cgaggacttc cgcatg 26 13 26 DNA Artificial SequenceDescription of Artificial Sequence Synthetic DNA 13 ccggatccagggctgagtta aaggag 26 14 29 DNA Artificial Sequence Description ofArtificial Sequence Synthetic DNA 14 ccggatccga ggattccatt gtcttcctg 29

What is claimed is:
 1. A monoclonal antibody produced by hybridoma 7F5or 10A3, deposited as FERM BP-5711 and FERM BP-5713, respectively,wherein the monoclonal antibody specifically recognizes human L-PGDS. 2.A monoclonal antibody produced by hybridoma 9A6, deposited as FERMBP-5712, wherein the monoclonal antibody specifically recognizes humanL-PGDS.
 3. A monoclonal antibody produced by hybridoma 6F5, deposited asFERM BP-5710, wherein the monoclonal antibody specifically recognizeshuman L-PGDS.
 4. A monoclonal antibody produced by hybridoma 1B7,deposited as FERM BP-5709, wherein the monoclonal antibody specificallyrecognizes human L-PGDS.
 5. A method for detecting L-PGDS comprisingcontacting L-PGDS with the monoclonal antibody of any of claims 1, 2, 3or
 4. 6. A hybridoma selected from the group consisting of 7F5 or 10A3,deposited as FERM BP-5711 and FERM BP-5713, respectively.
 7. Thehybridoma 9A6, deposited as FERM BP-5712.
 8. The hybridoma 6F5,deposited as FERM BP-5710.
 9. The hybridoma 1B7, deposited as FERMBP-5709.
 10. A kit comprising one or two monoclonal antibodies thatspecifically recognize human L-PGDS, wherein the monoclonal antibodiesare selected from the group consisting of: (a) a monoclonal antibodyproduced by hybridoma 7F5 or 10A3, deposited as FERM BP-5711 and FERMBP-5713, respectively; (b) a monoclonal antibody produced by hybridoma9A6, deposited as FERM BP-5712; (c) a monoclonal antibody produced byhybridoma 6F5, deposited as FERM BP-5710; and (d) a monoclonal antibodyproduced by hybridoma 1B7, deposited as FERM BP-5709, wherein at leastone of the monoclonal antibodies is labeled with an enzyme or isbiotinylated.
 11. A method of detecting L-PGDS, the method comprisingcontacting the L-PGDS with one or two monoclonal antibodies thatspecifically recognize human L-PGDS, wherein the monoclonal antibodiesare selected from the group consisting of: (a) a monoclonal antibodyproduced by hybridoma 7F5 or 10A3, deposited as FERM BP-5711 and FERMBP-5713, respectively; (b) a monoclonal antibody produced by hybridoma9A6, deposited as FERM BP-5712; (c) a monoclonal antibody specificallyrecognizing human L-PGDS produced by hybridoma 6F5, deposited as FERMBP-5710; and (d) a monoclonal antibody specifically recognizing humanL-PGDS produced by hybridoma 1B7, deposited as FERM BP-5709, wherein atleast one of the monoclonal antibodies is labeled with an enzyme or isbiotinylated.